The long-term goal of this research project is to determine the pathophysiology underlying the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) and to identify drug targets that lead to effective ALS treatments. ALS is a debilitating and fatal neurodegenerative disease affecting upper and lower motor neurons, with a prevalence in the United States of 1 in 25,000 persons and an annual deficit to the economy of $256 to $433 million. Though studied for over two decades since the first ALS causing genetic mutation was discovered, we have yet to uncover the pathological processes that lead to progressive degeneration of motor neurons in ALS, or to develop effective treatments. However, years of research have revealed that excitotoxicity likely plays a role in ALS pathogenesis, and recent studies have shown that increased spontaneous action potential firing (activity) observed in in vivo ALS-model motor neurons is a consequence of abnormalities in spinal circuitry, though the underlying pathways have yet to be elucidated. Here, we show that motor neurons generated from mouse embryonic stem cells exhibit a similar phenotype of hyperactivity, and, mimicking the in vivo model system, the underlying cause is due to synaptic aberrations. Intriguingly, despite being hyperactive, we find the ALS-model motor neurons to be intrinsically hypoexcitable, likely due to compensatory homeostatic mechanisms. Preliminary data suggest that cell-autonomous processes that alter synaptic connectivity mediate these observed irregularities in our in vitro ALS-model system. Therefore, we hypothesize that either altered ratios of excitatory to inhibitory synapses, increased excitatory post-synaptic response, or a combination of both underlie the increase in spontaneous activity observed in ALS-model motor neurons. Uncovering these pathways is paramount to future mechanism-based drug discoveries that normalize motor neuron physiology. The objective of this proposal is to use an established stem cell model to elucidate mechanisms in ALS motor neurons that drive hyperactivity, by examining the types, relative prevalence, and efficacy of synapses. I will accomplish this objective with the following two aims: 1) Examine post-synaptic currents in wild type and ALS-model motor neurons plated on astrocyte microislands using whole-cell patch clamp and 2) Compare the density of ionotropic synaptic input in ALS model motor neurons to WT using immunocytochemical and electrophysiological analyses. The results of these analyses will uncover pathways that may underlie excitotoxic pathophysiology in ALS, and will further efforts toward our long-term goal of effectively treating ALS.